Proteins encoded by the ras proto-oncogene act as molecular switches responding to growth stimuli and signaling to the intracellular machinery the occurrence of an extracellular event such as binding of a growth hormone to a growth hormone receptor molecule. Binding of the hormone to its receptor (the external signal) switches the ras protein to the "on" position characterized by exchange of ras bound GDP for GTP. The tightly bound GTP in turn stimulates downstream target proteins, ultimately triggering a cascade of reactions leading to specific gene transcription and ultimately cell division [Barbacid, M., Ann. Rev. Biochem. 56:779 (1987), McCormick F., Nature 363:15-16 (1993)]. The normal (i.e. non-transformed) ras protein eventually switches to the off position by hydrolyzing bound GTP to GDP and the cell is poised to receive the next external signal.
Mutations to the ras proto-oncogene translate into amino acid substitutions in the GTP binding domain, activating the ras protein (p21.sup.ras) and biasing this molecular switch in the "on" position. Thus, the ras transformed cell behaves like a cell with a faulty switch, signaling extracellular hormone binding when none is present. Cells transformed in this way grow and differentiate in an abnormal way.
Transforming ras genes are the oncogenes most frequently identified in human cancers. Clinical investigations have identified activated ras genes in a wide variety of human neoplasms, including carcinomas, sarcomas, leukemias, and lymphomas. It is estimated that 40% of all human colon cancers and 95% of human pancreatic cancers contain activated ras oncogenes [Kuzumaki, N. Anticancer Res., 11:313-320 (1991)].
Recently, it has been discovered that the ras protein must be properly posttranslationally modified before it can function as a molecular switch. Stable modification of the carboxy terminus of ras proteins appears to be essential for correct localization within the cell membrane so that extracellular signals for cell growth and differentiation can be correctly passed along to the intracellular messengers. The ras proteins are posttranslationally modified by farnesylation of a cysteine residue located four residues from the carboxy terminus, followed by proteolytic cleavage of the three following amino acid residues and methylation of the free cysteine carboxyl. The farnesylation reaction is catalyzed by a 94 kda heterodimeric Zn.sup.2+ metalloenzyme, farnesyl:protein transferase, which transfers the farnesyl group, a 15 carbon isoprenoid lipid derived from mevalonate (a cholesterol precursor), from farnesyl pyrophosphate to the carboxy terminus cysteine sulfur of ras forming a stable thioether linkage. The farnesyl:protein transferase recognizes the ras carboxy terminus consensus sequence, CAAX, where the cysteine (C) is followed by two aliphatic (A) amino acids (usually valine, leucine, or isoleucine) and any amino acid X (including methionine). This consensus sequence or motif is frequently referred to as the "CAAX box" and is found in other ras related GTP-binding proteins such as fungal mating factors, nuclear lamins, the gamma subunit of transducin, rhodopsin kinase, and the alpha subunit of cGMP-phosphodiesterase.
Surprisingly, this enzyme does not require intact ras protein for transferase activity and can utilize tetrapeptides with the CAAX motif as substrates (Reiss et al., Cell, 62: 81-88 (1990)). This observation suggested that small tetrapeptides like CAAX or nonpeptide analogs thereof could compete with p21.sup.ras for the active site of the transferase and therefore might be of therapeutic utility.
Previously, it had been observed that mutation of the cysteine in the CAAX carboxy sequence of p21.sup.ras to serine prevented farnesylation, proteolysis, and methylation (Hancock, J. et al., Cell 57:1167-1177 (1989); Reiss et al., PNAS, 88:732-736 (1991)). Additionally, cells incubated with an inhibitor of mevalonate synthesis prevented ras farnesylation and the cells were no longer capable of cell division (Schafer et al., Science, 245: 379-385 (1989)).
These results, taken together, suggest that inhibition of farnesyl:protein transferase with peptides containing the CAAX motif would prevent farnesylation of p21.sup.ras and block the ability of ras to transform normal cells to cancer cells. (see e.g. EP 0 461 869 A2, EP 0 496 162 A2, EP 0 523 873, and EP 0 520 823). Thus it is believed that intracellular delivery of peptides having the CAAX motif to transformed cells would be an effective anti-neoplastic therapy.
Generally, however, small linear peptides do not make good therapeutics because of their susceptability to proteolysis, oxidation, and lack of transportability across cell membranes. Accordingly, a need exists for a stable and potent non-peptidyl farnesyl:protein transferase inhibitor that is permeable to cell membranes.
Recently, several non-peptidyl ras farnesyl transferase inhibitors were identified through microbial screening. Several antibiotics (UCF1-A through UCF1-C) structurally related to manumycin inhibited growth of Ki-ras-transformed fibrosarcoma [Hara, M., et al. Proc. Natl, Sci. USA, 90:2281-2285 (1993)]. ##STR2## These inhibitors are reported to have potential application in cancer therapy.
Burk, R., et al. WO 92/20336(Merck) also describe nonpeptidyl farnesyltransferase inhibitors prepared by modification of natural products having structures similar to the following compound: ##STR3## These compounds are reported to be useful in treating cancer, especially colorectal carcinoma, exocrine pancreatic carcinoma, and myloid leukemia.
Benzodiazepines and analogs thereof have been widely exploited as therapeutics, but have not been reported to be inhibitors of farnesylation of G-proteins such as p21.sup.ras. Benzodiazepines are well known as central nervous system (CNS) drugs effecting the neuro-inhibitory postsynaptic GABA receptor and chloride ionophore channel (see eg. Watjen et al., J. Med. Chem. 32:2282-2291]1989]). Benzodiazepine analogs have been employed as intermediates in the synthesis of various anti-HIV-1 compounds [see e.g. Kukla, M. J. et al., J. Med. Chem. 34:3187-3197 (1991)] and as antagonists of gastrin and cholecystokinin (CCK) [see e.g. EP 0 284 256, assigned to Merck, and Friedinger, Med. Res, Rev., 9 271 (1989)]. More recently, benzodiazepine analogs have been reported to be fibrinogen antagonists, inhibiting platelet aggregation [see e.g. WO 93/00095 assigned to SmithKline Beecham.]
It was therefore an object of this invention to identify nonpeptidyl compounds that more effectively antagonize farnesylation of low molecular weight G-proteins such as p21.sup.ras in disease states in animals, preferably mammals, and especially humans. It was a further object of this invention to identify compounds that inhibit isoprenylation of proteins in microorganisms, such as yeast and fungi, that produce disease states in plants or animals, preferably mammals, and especially humans. These and other objects of this invention will be apparent from consideration of the specification and claims as a whole.